Retroviral infections such as for example infections by the human immunodeficiency virus (HIV) are still one of the most important and most widespread human diseases.
With regard to AIDS, an estimated 39.5 million people are living with HIV, the retrovirus causing AIDS. Recent data show that even in 2006 there were about 4.3 million new infections with some areas in the world where the infection rates have risen by more than 50% since 2004. Further, in 2006, about 2.9 million people died of AIDS-related illnesses according to the AIDS Epidemic Update 2006, published by the WHO (in December 2006).
The primary goals of antiretroviral therapy applied are a reduction of HIV-related morbidity and mortality, an improvement of quality of life, a restoration and preservation of immunologic function, and maximal and durable suppression of the viral load. Today antiretroviral therapy, more precisely, treatment regimens against HIV primarily rely on virus enzyme inhibitors and molecules that inhibit virus-cell fusion.
In this regard there are currently four classes of anti-HIV medicines available that are used in the treatment of AIDS. These drug classes target specific steps in the HIV replication process.
Fusion inhibitors (FI) are the first class of active agents, which work outside of host cells in order to prevent HIV from fusing with, entering and infecting these cells. A related approach is to prevent binding of HIV to the target cell via the CD4 receptors and co-receptors called CCR5 or CXCR4 on the surface of the target cells.
The three other classes of active agents work inside the cell. The so-called nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI) are used to prevent the replication of the virus inside host cells after they have been infected with HIV.
Examples of NRTIs and NNRTIs preventing HIV from making a copy of its genetic information (thus producing the so-called proviral DNA) are 3TC (lamivudine, Epivir), abacavir (Ziagen), AZT (zidovudine, Retrovir), d4T (stavudine, Zerit), ddC (zalcitabine, Hivid), ddI (didanosine, Videx/VidexEC), FTC (emtricitabine, Emtriva), Efavirenz (Sustiva) and nevirapine (Viramune).
The PIs target the HIV enzyme protease involved in virus assembly. Examples for these active agents are Amprenavir (Agenerase), atazanavir (Reyataz), fosamprenavir (Telzir), indinavir (Crixivan), lopinavir, nelfinavir (Viracept), ritonavir (Norvir) and saquinavir (Invirase/Fortovase).
One type of combination therapy currently used involving the use of more than one active agent is the Highly Active Antiretroviral Therapy (HAART) targeting the viral reverse transcriptase, protease and fusion (GULICK et al., 1997; LALEZARI et al., 2003). The application of this therapy has resulted in transforming HIV-1 infection into a chronic illness that has curtailed the morbidity of infected individuals.
One drawback, however, of all of current treatment strategies is that they only suppress the viral life cycle without eradicating the infection. The major obstacle in such therapies appears to be the establishment of long-lived reservoirs of HIV-1, particularly in latently infected resting CD4+ T cells (CHUN et al., 1998; FINZI et al., 1997), requiring life-long HAART.
Unfortunately, in a growing number of patients long-term HAART is accompanied by significant adverse side effects including mitochondrial toxicity, lipodystrophy, diabetes mellitus and osteoporosis (DYBUL et al., 2002). Substantial drug toxicities often result in inadequate adherence, resulting in the suboptimal inhibition of virus replication. As a consequence, new strains of HIV-1 are emerging that are resistant to suppressive treatments (LITTLE et al., 2002). In view of the increasing number of resistant HIV strains new active agents are necessary and are currently under development. In addition, further viral targets and novel inhibition strategies are being tested for improved control of HIV-1 (DONZELLA, 1998; CHIU et al., 2005; HAZUDA et al., 2004; HAUBER et al., 2005).
An alternative approach discussed in the art is to target the provirus inserted into the genome of the host cell. Excision of the proviral DNA from the host's genome for example would prevent further HIV replication and differs from current methodologies in that it has the potential to eradicate even dormant virus present in the genome of the host.
One class of proteins that were considered for use in this alternative approach are site-specific recombinases (FLOWERS et al., 1997). Site-specific recombinases mediate a multitude of functions in nature from gene rearrangement to genome segregation, such as for example excision, inversion, or integration of defined DNA units (reviewed in STARK et al., 1992).
One of the simplest and best understood recombinases is the Cre recombinase from bacteriophage P1 that resolves genome dimers into monomers by recombination between two identical double-stranded DNA sites of a particular sequence (HOESS & ABREMSKI, 1985). The Cre recombinase has found widespread use in mouse genetics (NAGY, 2000). Cre is a 38 kDa protein that was named after is function, as it causes recombination (STERNBERG & HAMILTON, 1981). Prerequisite for this recombination is the alignment of two recombination sites recognised by Cre in antiparallel orientation which are then bound by four identical Cre subunits that join to form a ring in which each subunit contacts two adjacent subunits and one half site of one recombination site (HOESS & ABREMSKI, 1985). The recombination site recognised by Cre is a 34-bp doublestranded DNA sequence known as loxP (from locus of crossing over (x), P1; STERNBERG & HAMILTON, 1981), which is palindromic with the exception of its eight innermost base pairs (referred to as the spacer), which impart directionality to the site.
Some site-specific recombination systems, including the Cre/loxP-system function without accessory proteins or cofactors and function under a wide variety of cellular conditions. However, since the site-specific recombinases function through specific interactions of the recombinase enzyme subunits with their cognate DNA target sequences, the use of these enzymes is restricted by the requirement that the targeted DNA regions must contain appropriately positioned target sites (LEWANDOSKI, 2001). To date, no wild-type recombinase has been identified that recognises native retroviral sequences as their DNA target sequences.
Extensive mutational and structural analyses of site-specific recombinases have been carried out in recent years to alter their properties and to achieve a better understanding of the intricate mechanisms of these enzymes (for a review see VAN DUYNE, 2001; and COATES et al., 2005). A lot of studies focussed on the Cre recombinase to explore its evolvability. Several studies demonstrated that Cre target specificity could be altered when few nucleotides in its loxP recognition site were changed (BUCHHOLZ & STEWART, 2001; SANTORO & SCHULTZ, 2002; RUFER & SAUER, 2002). Further studies addresses the engineering of mutated loxP target sites containing sequences from the LTR of HIV-1 to develop possible target sites for the use of Cre as antiviral strategy (LEE & PARK, 1998; LEE et al., 2000). To date, however, it has not been possible to generate a recombinase that recognises native asymmetric HIV sequences as their DNA target sequences.
The method of directed evolution is a powerful method to select enzymes with altered specificities (reviewed in Yuan et al., 2005; and JOHANNES & ZHAO, 2006). In the beginning this method was used to isolate improved enzymes on the basis of RNA by selecting RNA molecules with altered substrate sites. The use of PCR-based method allows the screening of very large libraries and the recovery of successful coding regions from a pool of candidates. In the directed evolution of proteins, by contrast, the screening for and the recovery of improved mutants, which are identified by alterations in the properties of the protein, requires a method for retrieving the nucleic acid sequence encoding the protein. The link between the protein and its coding sequence has often been maintained by compartmentalisation. Consequently, library screening in directed protein evolution has been limited to “one-by-one” approaches that maintain the compartments, and the advantages associated with screening pools of candidates have not been available.
This limitation has been overcome by the development of methods that allow the crosslinking of proteins to their respective messenger RNAs (mRNAs) using mRNA-protein fusions and ribosome display. Functional screens for improved protein properties were thus coupled to direct retrieval of corresponding coding molecules, and large pools have been screened in vitro (see for example BUCHHOLZ et al., 1998). A further improvement of directed protein evolution was achieved by the so-called substrate-linked protein evolution (SLiPE; BUCHHOLZ & STEWART, 2001), wherein the substrate of the recombinase was placed on the same DNA molecule as the protein coding region. In this manner, when the recombinase was expressed within a compartment, its action altered the DNA substrate next to its own coding region. Consequently, a library could be screened as a pool by PCR to amplify only candidate coding regions that were next to an altered substrate. This allows the screening of large libraries conveniently for rapid retrieval of successful coding regions. This method was applied for altering the DNA specificity of Cre recombinase and adapting it to a new recognition target site (BUCHHOLZ & STEWART, 2001).
However, a decisive drawback of the use of any recombinase for the excision of retroviral DNA is the need of the recombinase for symmetric target sites, which are typically not found at least twice in the proviral DNA to allow the use of present recombinases.
In view of the potential of site-specific recombinases and the need of finding an AIDS therapy eradicating HIV-1 provirus from the genome of host cell, the problem underlying the present invention thus resides in providing a method for preparing a tailored recombinase, which tailored recombinase recombines asymmetric target sites within the LTR of proviral DNA of a retrovirus inserted into the genome of a host cell thus excising the provirus from the genome of the host cell.